Abstract
砷、 鎘、 鎳，已被國際癌症研究中心認定為人類的致癌物。這些金屬都會抑制核酸修補，而這可能與致癌機轉有關。這些金屬均會與巰基結合。本論文研究探討的問題是：這三種金屬是否經由與巰基結合而抑制核酸接合酵素的作用？如果是，它們之間的差異又在那裡？另外，每一個重金屬最有效的解毒劑為何？在以中國倉鼠卵巢細胞 K1 細胞株的萃取液，測試核酸接合酵素，接合寡核甘酸活性的實驗中，我們使用一種鄰位雙巰基的鍵結劑 phenylarsine oxide (PAO)，以及一種單巰基甲基化劑 N-ethylmaleimide (NEM)，做為分辨單巰或雙巰結合的指標。實驗結果顯示: 對核酸接合酵素活性的抑制能力依序為: PAO > NEM > 氯化鎘 (Cd)> 氯化鎳 (Ni) > 亞砷酸鈉 (As)。除了 DTT 不能解除 Ni 的抑制作用，以及金屬硫蛋白 metallothionein(MT) 只能解除 Cd 的抑制作用外，其它的抑制作用均可被單巰基化合物，如 2-mercaptoethanol (2-ME) 或glutathione (GSH)，及雙巰基化合物，如 dithiothreitol (DTT) 或dimercaptosuccinic acid (DMSA) 等解除。DTT 較 2-ME 或 GSH 在解除 As 或 PAO 對核酸接合酵素活性的抑制上更為有效。丙酮酸脫氫酵素複合體在酵素活性區域上有臨位的雙巰基。DTT 和 DMSA 對這個酵素活性抑制作用的解除也較 2-ME 或 GSH 更為有效。這些結果顯示 As 及 PAO抑制核酸接合酵素活性是經由與蛋白質的臨位雙巰基結合而使酵素失去活性。但 2-ME、 GSH 及 DTT 解除 Cd 或 NEM 抑制丙酮酸脫氫酵素複合體活性的能力相當，此結果顯示 Cd 可藉由與蛋白質上的單巰基或雙巰基鍵結合抑制酵素的活性。在解除 Ni 對核酸接合酵素活性或丙酮酸脫氫酵素複合體活性抑制上 GSH 遠較 2-ME、DTT 及 DMSA 更為有效，這表示 Ni可能是經由鍵結單巰基而抑制酵素活性。在所有測試的含巰基化合物中，解除 As 抑制最有效的是 DMSA，解除 Cd 最有效的是 MT，解除 Ni 最有效的是 GSH，解除 NEM 最有效的是 DTT，而解除 PAO 最有效的是 DMSA。Arsenic, cadmium, and nickel are well-known human carcinogens.All these metals have been reported to inhibit DNA repair whichmay involve in carcinogenesis. They all have been reported tohave an affinity with thiol. The questions addressed in thisinvestigation are: Do they bind to enzyme thiols to inhibit DNAligation? If they do, then what is the difference? The resultsshowed that DNA ligation in CHO cell extract was sensitive to adithiol binding agent, phenylarsine oxide (PAO), and amonothiol binding agent, N-ethylmaleimide (NEM). The potencyorder of inhibition was PAO > NEM > cadmium chloride (Cd) >nickel chloride (Ni) > sodium arsenite (As). Inhibition of DNAligation by these agents all could be restored by monothiolcompounds such as 2-mercaptoethanol (2-ME), and glutathione(GSH), and ditiol compounds such as dithiothreitol (DTT) anddimercaptosuccinic acid (DMSA), except DTT could not reduce theinhibitory effect of Ni. DTT was more effective than 2-ME andGSH in reducing the As and PAO inhibition of ligation.Moreover, DTT and DMSA were also more effective than 2-ME andGSH in rescuing the As and PAO inhibition of the activity ofpyruvate dehydrogenase (PDHase), an As-sensitive enzymecontaining a vicinal dithiol at active site. These resultssuggest that As inhibits DNA ligation by binding to the dithiolgroups of DNA ligases. The results showing 2-ME, GSH and DTTwere equal potent in reducing Cd and NEM inhibition of PDHaseactivity, suggest Cd can bind both mono- and di-thiol groups toinhibit an enzyme activity. Whereas, the results showing GSHwas more effective than 2-ME, DTT and DMSA in rescuing Niinhibition of DNA ligation and the activity of PDHase, suggestthat Ni preferentially binds to monothiol residue to inhibitenzyme activity.